Studies On The Cytotoxic Effect And Polarization Of Macrophage Induced By Cypermethrin And Its Related Mechanism

Cypermethrin,a type ? pyrethroid pesticide,is widely used for control of agricultural and household pests.In recent years,due to the high toxic organophosphate and carbamate pesticides were prohibited to use,and pyrethroid pesticicide such as cypermethrin has high efficiency,broad spectrum for killing pests and acute low toxicity to mammals,the scope and dosage of its application were increased,which made environmental pollution increase sharply.The productive and non-productive poisoning cases of this kind of pesticide have been reported and its related toxicity mechanism has also been gradually deepened.It is well known that the immune response and immune function imbalance can cause a variety of diseases including infection,tumor and autoimmune diseases.There are previous reports about immune toxicity of cypermethrin.After mice exposed to cypermethrin,immune function circadian rhythm will disappear,which suggest immune system is the target of cypermethrin.Most of the researches show that the lethal dose of cypermethrin can inhibit humoral immunity and cellular immunity of animals.Macrophage as one of the important components of natural immune system,plays important role in the process such as inflammation,defense,repair,and metabolic physiology,and is also the key factor for maintaining stability.In order to illustrate the immune toxicity and its mechanism of cypermethrin more clearly,this study chose macrophages from mice as model,and discussed toxic effect of cypermethrin on macrophages and its effect on function of macrophages.1.Studies on the mechanisms of cytotoxic effect induced by cypermethrinFirst of all,our experiment reference the concentration range of cypermethrin on citrus pest spray:30?100 mg/L,and we choose 0-200?M(equivalent to 83.2 mg/L)of cypermethrin exposure to macrophages.Research shows that 100?M and 200?M cypermethrin exposure reduce cell viability using MTT assay compared to the control.We found that cypermethrin induced apoptosis in a dose-dependent manner by Fluorescence Activating Cell Sorter.We also found that cypermethrin induced hihger intracellular ROS than the control group,and especially the significant difference was found in 200?M exposure group.Moreover,cypermethrin was able to induce G1 cell cycle arrest and DNA damage.Pretreatment with antioxidant N-acetylcysteine(NAC)effectively protected cell against cytotoxicity induced by cypermethrin,such as:NAC treatment could increase cell viability,and NAC treatment could abrogated cypermethrin-induced cell DNA damage,apoptosis and G1 cell cycle arrest.In addition,cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase(JNK)and extracellular regulated protein kinases 1/2(ERK1/2)phosphorylation.NAC effectively inhibited JNK and ERK1/2 activation.ERK1/2 specific inhibitor PD98059 and JNK SP600125 specific inhibitors can effectively suppress the apoptosis induced by cypermethrin.2.Studies on the macrophage polarization and function induced by cypermethrin and its related mechanismMacrophage is highly plasticity and heterogeneity.In specific microenvironment or under stimulation in vivo and in vitro,macrophages can usually be activated into two phenotypes:the classic M1 type activated by IFN-gamma and LPS,and alternative actived into M2 type.M1 macrophages can eliminate intracellular pathogen,and protect against tumor cells,M2 macrophages produce a variety of immunosuppressive cytokine to promote cancer growth,inhibit inflammation and promote the tumor cell growth and metastasis.This part of the study is based on that cypermethrin dose not affect macrophage viability.And it mainly explore the impact of cypermethrin on macrophage polarization and function and the related mechanism.Treatment with cypermethrin can suppress the M1 type of inflammatory cytokines induced by LPS such as TNF-a,IL-6 and iNOS,suggesting that cypermethrin can suppress M1 macrophages polarization.We also find that cypermethrin will increase M2-related genes such as Arg-1,Fizzl and Mgl2 in a dose dependent manner.All the results show that cypermethrin can promote macrophage from M1 to M2 polarization.And cypermethrin induced M2 macrophages polarization,mainly by reducing the expression of miRNA-155.Interestingly,miRNA-155 overexpression significantly inhibited M2-associated genes induced by cypermethrin.In contrast,inhibition of miRNA-155 significantly increased M2-associated genes.And Bcl6 expression increased significantly induced by cypermethrin.Futhermore,to obtain evidence that Bcl6 is a direct target of miR-155,the wild-type 3'-UTR or a mutation of the 3'-UTR of Bcl6 were cloned into the vector to obtain the luciferase reporter constructs.By co-transfection of the luciferase reporter plasmids and miR-155 mimics into HEK293T cells,we found that miR-155mimics significantly inhibited the luciferase activity of cells transfected with wild-type Bcl63'-UTR vector,while the mutation Bcl6 3'-UTR vector has no obvious change.These results showed that Bcl6 can be directly modulated by miR-155.Thus,cypermethrin repressed expression of miR-155,which in turn enhanced Bcl6 expression.Inhibition o Bcl6 markedly reduced M2-associated genes such as Arg-1,Fizzl and Mgl2,while Bcl6 overexpression significantly increased M2-associated genes.These results showed that Bcl6 plays an important role in M2 polarization induced by cypermethrin.In addition,we also found that Bcl6 as a transcription inhibitor can inhibit the expression of MKK4.Overexpression of Bcl6 significantly suppressed MKK4 expression.As MKK4 is the activated kinase of JNK,JNK activation was also suppressed.Inhibition of Bcl6 significantly increased MKK4 expression,promoting its downstream JNK phosphorylation and activation.When miRNA-155 was overexpressed,inhibition of MKK4 or suppressing the activation of JNK could increase the M2-related genes while suppress M1-related genes compared with control.When miRNA-155 was inhibited,knockdown of Bcl6 was able to suppress M2-related genes while increased Ml-related genes compared with control.These results show that cypermethrin regulates macrophage from Ml to M2 polarization through the miRNA-155/Bcl6/MKK4/JNK pathway.In vitro and In vivo experiment,M2 macrophages induced by cypermethrin can significantly promote lung cancer cell migration and growth compared with the M1 macrophages induced by LPS,which suggesting cypermethrin has influence on macrophage function.Taken together,we can get the following conclusions from this research:(1)Cypennethrin exposure for 48h reduces cell viability in a dose-dependent manner.Cypermethrin can induce intracellular ROS,and pretreatment with NAC is able toabrogate DNA damage,G1 cell cycle arrest and apoptosis induced by cypennethrin.Cypermethrin activates JNK and ERK1/2 pathways,and induces apoptosis regulated by ROS-mediated JNK/ERK pathway.(2)Cypermethrin repressed expression of miR-155,which in turn enhanced Bcl6 expression,and thereby reduced MKK4 expression and subsequently inhibited JNK activation,making the macrophage from M1 to M2 type.At the same time,cypermethrin can enhance macrophage to promote tumor cell migration and growth.The results are able to give evidence for systematic evaluation of immune toxicity induced by cypermethrin.